In the sliding actin in vitro motility assay, the movement of fluorescently-labeled actin over a surface coated with myosin is visualized in the fluorescent microscope. In all the cases reported to date, the underlying myosin surface is not directly visualized. It is usually assumed that the bound myosin filaments or monomers are randomly oriented. We have isolated large native thick filaments from molluscan muscle using a rapid and gentle method. These thick filaments are between 10 and 50 um long depending on the source of the muscle and can be directly visualized on the glass surface using video-enhanced differential interference contrast microscopy. This allows us to directly correlate the movement of the fluorescently-labeled actin filaments with their position on the native bipolar thick filament. Our observations show that actin can travel both toward and away from the center of the thick filament and that the polarity of actin determines the direction of movement. Actin filaments moving toward the center of the thick filament travel about 9 times faster than those traveling away from the center. The movement of actin away from the center of the thick filament is opposite that which usually occurs in muscle contraction and suggest that the heads of myosin must be very flexible.